This project seeks to use genetic, biochemical and physiological studies to investigate the transport and metabolism of carbohydrates by oral bacteria. A. plasmid (PLZ64, 23M dalton) that encodes the lactose transport system (lactose phosphoenolpyruvate-dependent: phospnotransferase system; PEP:PTS) and the enzyme phospho-Beta-galactosidase has been isolated from Lactobacillus casei. A detailed physical map of the plasmid was constructed and the component restriction fragments cloned into Escherichia coli. The plasmid was found to have significant homology with plasmids isolated from other L. casei strains. A number of lactose plasmids isolated from L. casei were also found to contain sequences hybridizing to a cloned phospho-Beta-galactosidase gene probe. Several small, multi-copy, cryptic L. casei plasmids were mapped, and cloned into E. coli and Streptococcus sanguis. These chimeras are being modified for use as Lactobacillus cloning vectors. In order to establish a transformation system in the genus, methods to generate and regenerate protoplasts were developed. It was found that L. casei strains secrete DNAase; mutants lacking this enzyme were isolated. Cosmid clone banks containing whole genomic DNA isolated from several Lactobacillus and Streptococcus species were also prepared and are being screened for specific genes. Other studies characterized the lactose and galactose PEP:PTS in L. casei. The presence of a 2-deoxy-glucose (2-DG) mediated "futicle cycle" that uncouples growth and ATP production from metabolism and biosynthesis of PEP in S. lactis was proposed to be the major growth inhibitory effect of 2-DG on streptococci. The hexase 6-phosphate phosphohydrolase that cleaves intracellular 2-DG 6-phosphate was purified and characterized. The efflux of hexose analogs from cells is being studied through the use of lactose analogs in which 2-DG or 2-Deoxy-2-fluorogluse (2-FG) are substituted for the glucosyl moiety of lactose. These analogs were found to be substrates of the lactose PEP:PTS and were used to transport 2-DG and 2-FG into cells lacking the mannose PEP:PTS. Isolation of mannose PTS deficient mutants and their biochemical characterization is in progress.